Marine sponges are the focus of biological, chemical and ecological research aimed at understanding and exploiting their unique characteristics. As sessile benthic animals renown for their distinctly elementary metazoan organization, sponges are an exceptional target for studies of self recognition, immunity, and chemical ecology. Many are sources of pharmaceutically important compounds (Faulkner, D. J. 2000, Nat Prod Rep 17:1–6). Thus, controlled cellular and molecular studies in sponges present the opportunity to learn more about our multicellular condition, and to understand systems that are sources of compounds with human therapeutic value.
There have been numerous attempts to develop a classic in vitro model (i.e., a cell line) as a tool for marine sponge cell research. While some progress has been made, the ultimate goal of a clonal, axenic, continuously dividing marine sponge cell line has yet to be achieved. Despite this limitation, research is progressing with development of alternative culture systems (Custodio, M. R. et al. 1998, Mech Ageing Dev 105(1–2):45–59; Kreuter, M. H. et al. 1992, Comp. Biochem. Physiol. 101C(1):183–187; Munro M. H. et al. 1999, J Biotechnol 70(1–3):15–25; Muller, W. E. et al. 2000, J Ant Prod 63(8):1077–1081), reports of cell proliferation (Krasko, A. et al. 2002, DNA Cell Biol 21(1):67–80), and elucidation of some of the basic cellular and molecular traits of marine sponge cells (Muller, W. E. et al. 2001, Gene 276(1–2):161–173; Schutze, J. et al. 2001, J Mol Evol 53(4–5):402–415).
One advance is the discovery that marine sponge cells respond to the mitogenic lectin, phytohemagglutinin (PHA) (Pomponi, S. A. and R. Willoughby 1994 “Sponge cell culture for production of bioactive metabolites” In: van Soest, van Kempen, and Braekman, editors. Sponges In Time and Space. Rotterdam: Balkema. p 395–400). Previously, this sponge cell culture phenomenon has been documented only by noting cell numbers, protein content, esterase activity, and DNA content in primary cultures of PHA-treated cells (Willoughby, R. and S. A. Pomponi 2000, “Quantitative assessment of marine sponge cells in vitro: development of improved growth medium” In Vitro Cell Dev Biol—Animal 36:194–200). Even in mammalian cell lines, the molecular basis of the PHA response has been poorly understood, though some recent work has begun to identify some associated molecules.
The Model Sponge
The marine sponge Axinella corrugata (Phylum Porifera, Class Demospongiae, Order Axinellida, Family Axinellidae) (FIG. 1) has been used as a model system for more than ten years. It is relatively easy to collect and maintain, and produces the bioactive compound stevensine (Wright, A. E., S. E. Chiles, S. S. Cross 1991, J Nat Prod 54(6):1684–1686) (FIG. 2), which has antitumor properties (U.S. Pat. No. 4,729,996) and also functions as a neurotransmitter blocker (Coval, S. J. et al. 1996, U.S. patent application Ser. No. 08/644,138). The production of this compound, which is believed to be of sponge origin, makes A. corrugata an appropriate candidate for cell culture studies that focus on biosynthesis as a model for in vitro production of potentially therapeutic products (Andrade, P. et al. 1999, Tetrahedron Lett 40(26):4775–4778). Success in establishing primary cell cultures of this species (Pomponi, S. A., R. Willoughby, and M. Kelly-Borges 1997a, “Sponge Cell Culture” In: Cooksey K, editor. Molecular Approaches to the Study of the Ocean. Chapman & Hall. p 423–429; Pomponi, S. A. et al. 1997b, “Development of techniques for in vitro production of bioactive natural products from marine sponges” In: Invertebrate Cell Culture: Novel Directions and Biotechnology Applications. Maramorosch K, Mitsuhashi J, editors. Science Publishers, Inc. p 231–237; Pomponi, S. A. et al. 1998. “In vitro production of marine-derived antitumor compounds” In: Le Gal Y, Halvorson HO, editors. New Developments in Marine Biotechnology. New York: Plenum Press p 73–76) and in vitro production of stevensine (Pomponi et al. 1997b, 1998 supra) have been demonstrated. In addition, A. corrugata has been used as an in vitro model for the analysis of the effects of culture medium factors on DNA, protein, and esterase activity (Willoughby and Pomponi 2000 supra).
Marine Sponge Genes and Gene Expression
Previously, some insight into marine sponge potential for molecular response has been achieved by comparing individual sponge nucleic acid sequences to those of model organisms, thus accomplishing gene discovery by database homology analysis.
Many of these previous studies have focused on phylogeny and evolutionary genetics, rather than characterization of in vitro (or even in situ) physiology for functional purposes. Indeed, few have looked at the actual expression of the characterized genes, though a recent contrary trend is evident. In one of the earliest expression studies, Schroder et al. 1988 (J Biol Chem 263(31):16334–16340) used immunoprecipitation to quantify ras expression in marine sponge cells. Biesalski et al. 1992 (Oncogene 7(9):1765–1774) reported down-regulation of a myb-related gene in cells of Geodia cydonium. Also, Pfeifer et al. (1993b, J Cell Sci 106 (Pt 2):545–553) reported increased polyubiquitin expression in response to homologous aggregation factor. These studies employed dissociated cells and were therefore an early look at the function of sponge cells in vitro.
More recent studies, many of which utilize intact sponge tissue or re-aggregated cells, include those by Wiens et al. (2000b, J Mol Evol 50(6):520–531) and Kruse et al. (1999, J Cell Sci 112(part 23):4305–4313), who looked at differential expression in response to allograft rejection in marine sponge tissue. Profilin expression was also up-regulated in the presence of non-self sponge molecules (Muller, W. E. et al. 1999b, DNA Cell Biol 1(12):885–893). Potential self-recognition molecules were up-regulated in autografts according to Wimmer et al. (1999b, Cell Adhes Commun 7(2):111–1124), Fernandez-Busquets et al. (1998, J Biol Chem 273(45):29545–29553), and Blumbach et al. (1999, Immunogenetics 9(9):751–763.). Molecules associated with immune responses were reviewed by Muller et al. (1999c, Transplantation 68(9):1215–1227.). Scheffer et al. (1997, Biological J Linnean Soc 61:127–137) used whole sponges to study SRF expression in response to heat stress. Whole sponges were also used to document increased MA-3 expression (Wagner, C. et al. 1998, Mar Biol 131:411–421). Weins et al. (1999a, Tissue Cell 31(2):163–169) reported down-regulation of a putative tumor suppressor in response to cadmium exposure, and Krasko et al. (1999, J Biol Chem 274(44):1524–1530) reported up-regulation of a protein kinase and a potential ethylene-responsive protein in sponge tissues exposed to ethylene. Utilizing intact tissue, Weins et al. (1999c, Marine Biol 133:1–10) documented increased HSP70 and thioredoxin expression in response to 17β-estradiol. Increased HSP70 expression was also noted in response to tributyltin (Batel, R. et al. 1993 Mar Ecol Prog Ser 93:245–251.). Phosphorylation of p38 was detected in sponge primmorphs treated with hypertonic medium (Bohm, M. et al. 2000, Biol Cell 92:95–104). A similar culture system was used to study differential expression of a longevity assurance-like gene (Schroder, H. C. et al. 2000 Mech Devel 95:219–220) as well as collagen and silicatein genes (Krasko, A. et al. 2000 Eur J Biochem 267:4878–4887.). Actual cell cultures (not tissue or primmorphs) were once again used to demonstrate ras up-regulation in response to sponge aggregation factor by Wimmer et al. (1999b, Cell Adhes Commun 7(2):111–1124.). Intact sponges stressed by exposure to UV light demonstrated increased expression of an excision repair gene homologue as measured by Northern blot comparisons (Batel, R. et al. 1998 Mutat Res 409(3):123–33.).
Recently, researchers have begun to directly explore sponge functional genetics in relation to that of other organisms. Muller and colleagues have begun to present multiple cases for genetic homology, as well as functional similarities, between sponges and higher organisms (Muller, W. E. et al. 2001, Gene 276(1–2):161–173; Gamulin, V. et al. 2000, Biological Journal—Linnean Society 71( ):821–828; Seack, J. et al. 2001, Biochim Biophys Acta 1520(1):21–34; Bohm, M. et al. 2000, Biol Cell 92:95–104; Wiens, M. et al. 2000a, Cell Death Differ 7(5):461–469; Pahler, S. et al. 1998c, Proc R Soc Lond B Biol Sc. 265(1394):421–425).
DNA Microarray Technology
DNA microarray technology is relatively new, and is of great interest to the biology community due to its power to simultaneously analyze gene expression for thousands of genes. It offers a functional means to begin to resolve some of the complexities of regulation in biological systems. The technique is based on hybridization of complementary DNA molecules on two-dimensional surfaces upon which thousands of oligonucleotides or DNA fragments (probes) are attached, thus facilitating the simultaneous screening/hybridization of thousands of probes and thousands of targets (Ramsay, G. 1998 Nature Biotechnology 16:41–44.).
Since no sponge DNA array currently exists, labeled sponge target molecules were applied to an existing array of human gene sequences. The system uses nylon microarrays and radioactive detection. Since the identities of the probes are known, they provide indications of the identities of the hybridizing sponge gene sequences.
Phytohemagglutinin
Phytohemagglutinin was known for some time as simply a T lymphocyte mitogenic activator (Robbins, J. H. 1964, Experientia 20(3):164–168.). More recently, it has been drawn into the explosion of gene expression research following the development of powerful technologies such as microarray analysis. It has now been shown to have mitogenic effects in a number of cell types, including intestinal epithelia (Otte, J. M. et al., 2001, Digestion 64(3):169–178.) and fibroblasts (Mustafa, M. et al. 2000, Cytokine 12(4):368–373). The details of the genetic response to PHA are beginning to emerge in greater complexity beyond the well-known and long-observed cytokine production response (Janefjord, C. K and M. C. Jenmalm M C. 2001, Clin Exp Allergy 31(10):1493–1500; Beppu, R. et al. 2001, Immunol Invest 30(2):143–156). The complexity of the immune response is suggested by the finding that PHA stimulation elevates serotonin receptor mRNA levels (Abdouh, M. et al. 2001, J Biol Chem 276(6):4382–4388). Broad physiological effects such as elevations in ion transport mRNAs have also been reported (Vereninov, A. A. et al. Cell Physiol Biochem 11(1):19–26). Levels of c-fos and c-jun mRNA were elevated 30 minutes after PHA treatment of human lymphocytes (De Palma, L., E. Brown, and R. Baker 1998, Vox Sang 75(2):134–138.).
PHA stimulates intracellular signaling pathways related to production of cytokines and cell proliferation. Though details of its action in sponge cell cultures are unknown, it is associated with elevated sponge cell numbers in vitro (Pomponi, S. A. et al. 1997b “Development of techniques for in vitro production of bioactive natural products from marine sponges” In: Invertebrate Cell Culture: Novel Directions and Biotechnology Applications. Maramorosch K, Mitsuhashi J, editors. Science Publishers, Inc. p 231–237).
Receptors. PHA can function in concert with other stimulatory agents and its effects can vary qualitatively according to its concentration (Modiano, J. F. et al. 1999, Cell Immunol 197(1):19–29). In addition, multiple isoforms of PHA exist, some of which exhibit different activities in certain cell types (Rebbaa, A. et al. 1996, J Neurochem 67(6):2265–2272). The exact role of PHA in receptor activation is still being elucidated. It has been shown to directly bind the epidermal growth factor (EGF) receptor, though details of its function at this site are not clear. Although receptor binding was demonstrated, PHA abrogated expected phenotypic events dependent on EGF receptor signaling in a human cell line (Rebbaa et al. 1996 supra). PHA seems to mimic effects of agents known to function via receptor protein-tyrosine kinases as well as G protein-coupled receptors. It has been shown to modulate both the expression and activity of G protein-coupled receptors (Consorzio et al. 1995; De Blasi A. et al. 1995, J Clin Invest 95(1):203–210). The monomeric G protein Ras is activated by PHA, resulting in stimulation of a signaling pathway known to promote T cell proliferation (Downward, J. et al. 1990, Nature 364(6286):719–723) and to participate in promotion of interleukin 2 production (Ohtsuka, T., Y. Kazario, and T. Satoh 1996, Biochim Biophys Acta 1310(2):223–232). Thus, activation of a variety of receptors by PHA can result in progression of multiple intracellular signals.
Signal Transduction. Specific effects of PHA are consistent with its role in promotion of cell replication and the cell cycle. Induction of immediate-early gene transcription via the AP-1 transcription factor is evident in PHA-induced up-regulation of fos and jun following PHA treatment of lymphocytes (Bulanova, E. G. 1997, Biochemistry 62(9):1021–1025). PHA has also been shown to promote mitogen-activated protein kinase activity and G1-phase cyclin-dependent kinase activation (Modiano et al. 1999, Cell Immunol. 197(1):19–29). Interleukin 2 production is a well-known result of PHA stimulation in lymphocytes (Mills, G. B. et al. 1990, J Cell Physiol 142(3):539–551).
Phospholipid and calcium signals. PHA is known to cause elevations of intracellular free Ca2+ in the form of peaks, plateaus, or oscillations associated with initial internal mobilization of calcium from intracellular stores and subsequent influx from outside the cell (Maltsev, V. A. et al. 1994, Immunol Lett. 42(1–2):41–47). SH2-type protein tyrosine kinases as well as G-type receptors phosphorylate phospholipase C (PLC) and result in PLC translocation to the cell membrane, initiating a series of intracellular events related to control of cell proliferation (Cooper, G. M. 1997, “The Cell: A Molecular Approach” ASM Press and Sinauer Associates, Inc. 673). Subsequent hydrolysis of phosphatidylinositol 4,5-biphosphate (PIP2) produces diacylglycerol, resulting in activation of protein kinase C and subsequent activation of MAP kinase cascades and/or translocation of NF□B to the nucleus, where it can mediate transcription of proliferation-associated genes (Kirillova, I., M. Chaisson and N. Fausto 1999, Cell Growth Differ 10(12):819–828) as well as genes involved in apoptosis (Kuhnel, F. et al. 2000, J Biol Chem 275(9):6421–6427). PIP2 hydrolysis also yields inositol 1,4,5-triphosphate (IP3), which prompts the release of stored Ca2+ from the endoplasmic reticulum, initiating a cascade of calcium signaling maintained by calcium influx from outside the cell (Hess, S. D., M. Oortgiesen, and M. D. Cahalan 1993, J Immunol 150(7):2620–2633). Sufficient increases in cytosolic calcium result in activation of calmodulin, which in turn activates a variety of proteins including kinases. In concert with calmodulin, calcium and calcineurin B activate the protein phosphatase calcineurin A, resulting in nuclear translocation of the nuclear factor of activated T cells (NFAT), facilitating secretion of interleukin 2 (an autocrine promoter of proliferation) (Mills, G. B. et al. 1990, J Cell Physiol 142(3):539–551; Baldari, C. T. et al. 1991, J Biol Chem 266(28):19103–19108) and coordination with other transcription factors regulating proliferation (Crabtree, G. R. 1999, Cell 96:611–614).
Calcineurin B. Calcineurin B is a regulatory sub-unit that is highly conserved among eukaryotes (Rusnak, F. and P. Mertz 2000, Physiol Rev 80(4):1483–1521). Indeed, the amino acid sequences for human and bovine calcineurin B are identical (Nargang, C. E., D. A Bottorff and K. Adachi 1994, DNA Seq 4(5): 313–318). Along with calcium and calmodulin, calcineurin B activates the catalytic subunit, calcineurin A (Sugiura, R, S. O. Sio, H. Shunto and T. Kuno 2001, Cell Mol Life Sci 58:278–288). Activated calcineurin participates in regulatory functions in multiple cellular processes, including translocation of transcription factors to the nucleus (Masuda, E. S. et al. 1998, Cell Signal 10(9):599–611) and control of mitosis (Mizunuma, M. et al. 1998, Nature 392(6673):303–306). Differing roles have been observed in mammals, yeasts, and even scallops (Uryu, M. et al. 2000, J Biochem 127:739–746). Calcineurin activation is associated with binding of calcium, while calcium elevations are associated with PHA treatment (Orie, N. N. W. Zidek and M. Tepel 1999, Exp Physiol 84(3):515–520). In yeast, calcineurin is a requirement for a calcium-induced G2 delay (Mizunuma, M. et al. 1998, Nature 392(6673):303–306). In an inverse scenario, increased expression of calcineurin was associated with reduced proliferation in leukemic cells (Kihira, H. et al. 1998, Int J Oncol 12(3): 629–634 and Omay, S. B. et al. 1996, Blood 87(7):2947–2955) Calcineurin B mRNA levels peak during differentiation of flagellate amoebas (Remillard, S. P. et al. 1995, Gene 154(1):39–45). Plant calcineurin B-like protein exhibits increased transcription in response to stress (Kudla, J. et al. 1999, Proc Natl Acad Sci USA 96(8):4216–4218). Additional studies implicate calcineurin as a major factor in the execution of apoptotic signals (Springer, J. E. et al. 2000, J Neurosci 20(19):7246–7251; Saito, S. et al. 2000, J Biol Chem 275(44):34528–34533; Tombal, B. et al. 2000, Prostate 43(4):303–317; Jayaraman, T. and A. R. Marks 2000, J Biol Chem 275(9):6417–6420; Asai, A. et al., 1999, i 274(48):34450–34458).